High resolution mapping of a novel non-transgressive hybrid susceptibility locus in barley exploited by P. teres f. maculata

Hybrid genotypes can provide significant yield gains over conventional inbred varieties due to heterosis or hybrid vigor. However, hybrids can also display unintended negative attributes or phenotypes such as extreme pathogen susceptibility. The necrotrophic pathogen Pyrenophora teres f. maculata (Ptm) causes spot form net blotch, which has caused significant yield losses to barley worldwide. Here, we report on a non-transgressive hybrid susceptibility locus in barley identified between the three parental lines CI5791, Tifang and Golden Promise that are resistant to Ptm isolate 13IM.3. However, F2 progeny from CI5791 × Tifang and CI5791 × Golden Promise crosses exhibited extreme susceptibility. The susceptible phenotype segregated in a ratio of 1 resistant:1 susceptible representing a genetic segregation ratio of 1 parental (res):2 heterozygous (sus):1 parental (res) suggesting a single hybrid susceptibility locus. Genetic mapping using a total of 715 CI5791 × Tifang F2 individuals (1430 recombinant gametes) and 149 targeted SNPs delimited the hybrid susceptibility locus designated Susceptibility to Pyrenophora teres 2 (Spt2) to an ~ 198 kb region on chromosome 5H of the Morex V3 reference assembly. This single locus was independently mapped with 83 CI5791 × Golden Promise F2 individuals (166 recombinant gametes) and 180 genome wide SNPs that colocalized to the same Spt2 locus. The CI5791 genome was sequenced using PacBio Continuous Long Read technology and comparative analysis between CI5791 and the publicly available Golden Promise genome assembly determined that the delimited region contained a single high confidence Spt2 candidate gene predicted to encode a pentatricopeptide repeat-containing protein. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05303-1.


Introduction
Hybrid barley was one of the first commercially available small grain hybrid crops in the world which have been grown since the 1980s [1].Currently, hybrid winter feed barley is grown on more than 200,000 ha in Europe [1].Thus, hybrid barley may also be a solution to boost malt barley yields to satisfy the demands of the brewing and distilling industries as climate change is predicted to cause world malt barley shortages [2,3].These models utilizing future climate scenarios predict that some barley growing regions will experience extreme heat and drought while other production regions will experience increased temperatures and excess moisture contributing to conditions conducive to disease development impacting yield and quality [3].For some host-pathogen genetic interactions, the use of hybrid barley may increase disease resistance [4], however, in other pathosystems, hybrids may reduce resistance or increase susceptibility.The outcome of these interactions also depends on the virulence mechanisms of the pathogen and the nature of the pathogen in terms of acquiring nutrients from the host, either as biotrophic or necrotrophic pathogens.
Plant-pathogen genetic interactions that determine compatible (susceptible) -vs-incompatible (resistant) interactions, typically rely on resistance (R) genes that encode receptor-like proteins/kinases (RLPs/RLKs) or nucleotide binding-leucine rich repeat (NLR) class proteins that recognize pathogen effectors or their action [5].However, diverse coevolution that occurs in pathosystems can also evolve into non-dogmatic interactions that do not conform to this typical mechanism such as the first cloned resistance gene, Hm1, which encodes a reductase that neutralizes a toxin [6].Additionally, increased characterization of resistance genes has identified novel R-protein classes that may or may not include integrated domains that follow the proposed integrated sensory or decoy domain model such as ankyrin-repeat domains or WRKY domains [7][8][9][10].WRKY transcription factors have already been implicated in resistance with HvWRKY6 required for Pyrenophora teres f. teres (Ptt) resistance in barley [11].Therefore, these studies showed that alternative mechanisms and classes of genes/proteins are involved in plant resistance responses and non-canonical candidate genes should be thoroughly investigated.
Susceptibility genes and their respective proteins are manipulated by necrotrophic fungal pathogens to facilitate disease development and have often initially been characterized as recessive resistance genes.The first confirmed necrotrophic pathogen susceptibility gene identified was Pc-2/Vb, conferring dominant resistance to oat crown rust and dominant susceptibility to Victoria blight [12].This was the first study showing that necrotrophic pathogens can hijack dominant resistance genes to elicit programmed cell death (PCD) responses, referred to as the hypersensitive response (HR).These PCD/HR responses provide effective resistance against biotrophic pathogens that require living host tissue to acquire nutrients by sequestering the biotrophic pathogen to small foci of dead cells.However, when these mechanisms are elicited by necrotrophic effectors (NEs) secreted by necrotrophic pathogens that require dead or dying tissue to acquire nutrients, the PCD response results in host susceptibility and continued pathogen proliferation [13].
In the barley-Ptt necrotrophic pathosystem (causal agent of net from net blotch), the dominant susceptibility locus Spt1 was first identified as the recessive resistance loci rpt.r and rpt.k in Rike and Kombar, respectively [14,15].The Spt1 locus was hypothesized to be two tightly linked genes providing recessive resistance to different Ptt isolates.However, high-resolution mapping led to the hypothesis of a single gene allelic series that confers isolate specific Spt1-mediated susceptibility [16,17].In addition, alleles of this same gene may also correspond to the dominant Rpt5mediated broad-spectrum resistance that mapped to the same region in CI5791 [17,18].These types of complex genetic interactions of susceptibility and/or resistance genes could have relevant effects on disease development when hybrids are deployed depending on the combinations of alleles and effector repertoires of pathogen isolates challenging the plant.
Pyrenophora teres f. maculata (Ptm) recently diverged from the close relative Ptt and is the causal agent of the barley disease spot form net blotch (SFNB) [19,20].SFNB is present worldwide and can cause significant yield losses, typically between 10 and 40%, but can also result in complete crop failure or degraded seed quality [21][22][23].To date, over 150 quantitative trait loci (QTL) have been mapped within the host of the barley-Ptm pathosystem [17], and seven regions within the wheat-Ptm pathosystem [24].The Ptm isolate 13IM8.3, is avirulent on CI5791 and Tifang, but hyper virulent on all CI5791 × Tifang F 1 individuals.Thus, the objective of this study was to determine the genetic components contributing to this non-transgressive hybrid susceptibility within barley.We therefore, high-resolution mapped the locus designated Susceptibility to Pyrenophora teres 2 (Spt2) locus using Ptm isolate 13IM8.3 to an ~ 198 kb region containing a single high confidence gene model predicted to encode a pentatricopeptide repeat-containing protein (PPR) based on the cultivar Morex V3 genome assembly.To our knowledge, the Spt2 locus is the first reported case of hybrid susceptibility within the same species, as opposed to hybridization of two closely related species.The Spt2-mediated susceptibility to Ptm isolate 13IM8.3 is non-transgressive, similar to heterosis, as only heterozygous individuals at the Spt2 locus are susceptible and is therefore not genetically stable [25].This phenomenon is also not the result of post-zygotic genetic incompatibilities such as hybrid necrosis that involves epistatic interaction between multiple loci and occurs in the absence of the pathogen [26,27].

Disease ratings
The parental accessions CI5791, Tifang and Golden Promise had mean disease rating scores of 1.25, 2.75 and 2.0, respectively, whereas F 1 accessions were universally susceptible with a mean disease rating score of 4 to 5 (Fig. 1).For the 9K derived CI5791 × Tifang genetic mapping, a total of 106 F 2 individuals were utilized, of which 63 were considered susceptible and 43 were considered resistant (Supplemental Information 2, Supplemental Fig. 2).For the 50K derived CI5791 × Tifang genetic mapping, a total of 340 F 2 individuals were utilized, of which 139 were considered susceptible and 201 were considered resistant (Supplemental Information 4, Supplemental Fig. 2).For the exome derived CI5791 × Tifang genetic mapping, a total of 609 F 2 individuals were utilized, which consisted of 285 susceptible and 324 resistant (Supplemental Information 6, Supplemental Fig. 2).For the genotypeby-multiplex sequencing (GMS) CI5791 × Golden Promise genetic mapping a total of 83 F 2 individuals were utilized, which consisted of 40 susceptible and 43 resistant (Supplemental Information 7, Supplemental Fig. 2).A chi-squared goodness-of-fit test was conducted to determine if the populations followed a 1:1 ratio.The populations used utilizing 9K, exome and GMS marker panels had p values of 0.05, 0.11 and 0.74, indicating they do not deviate from the expected ratio.The population utilizing the 50K marker panel had a p value of 0.0007, indicating the population does deviate from the 1:1 ratio.However, if only 4% (13 accessions) were within the opposing class, the population would not deviate from the 1:1 ratio.

Genetic mapping of CI5791 × Tifang with SNP markers derived from the 9K SNP array
The initial genetic mapping of Spt2 was conducted with the phenotyping of 106 CI5791 × Tifang F 2 individuals that were genotyped with a 365 PCR-GBS SNP marker panel resulting in 119 high quality polymorphic SNP markers spread across the barley genome.The QTL mapping detected a single highly significant locus on chromosome 5H delimited by the markers 11_10641 and 11_20236 with the most significant marker 12_20350 giving a LOD score of 23.1 (Figs.2A and 3A, Supplemental Fig. 1).LOD thresholds were calculated at 3.788 (α = 0.05) and 4.701 (α = 0.01).The 16.7 cM genetic interval (Fig. 3A) corresponded to 23.1 Mb of genome sequence from the Morex V3 reference genome assembly containing 285 annotated genes.

High-resolution genetic mapping of CI5791 × Tifang with SNPs derived from the 50K SNP array
Within the genomic interval initially delimited via genetic mapping a total of 22 new SNP markers were identified as polymorphic between CI5791 and Tifang using the 50K iSelect genotyping platform.The first round of high-resolution mapping utilized a total of 340 CI5791 × Tifang F 2 individuals (680 recombinant gametes) that were further saturated by successful amplification of an additional 20 PCR-GBS SNP markers from the 22 SNPs identified as polymorphic between the two parental lines.QTL analysis detected a highly significant locus on chromosome 5H between markers 12_30080 and JHI-Hv50k-2016-308620 with the most significant marker JHI-Hv50k-2016-308229 giving a LOD score of 36.0 (Figs.2B and 3A).LOD thresholds were calculated at 2.63 (α = 0.05) and 3.401 (α = 0.01).The 38.9 cM genetic interval (Fig. 3) corresponds to a 5.1 Mb genomic interval within Morex V3 reference genome assembly with 64 annotated genes.

High-resolution genetic mapping of CI5791 × Tifang with exome capture derived SNPs
The next round of high-resolution mapping utilized 609 individuals and 8 additional PCR-GBS markers derived from exome capture sequencing to saturate the genomic interval identified from 50K derived high-resolution mapping.One major QTL was detected within the chromosome 5H genomic interval between markers 5H_490859253 and 5H_492358011 with a peak LOD score of 85.4.LOD thresholds were calculated at 1.599 (α = 0.05) and 2.399 (α = 0.01).The 1.2 cM genetic interval (Fig. 3A) corresponds to a 1.5 Mb genomic interval within the Morex V3 reference genome assembly with nine annotated genes.

Genetic mapping of CI5791 × Golden Promise with genotyping-by-multiplex sequencing
The CI5791 × Golden Promise population consisted of 83 F 2 individuals and 180 markers spread across the barley genome, with 23 anchored to chromosome 5H of the Morex V3 reference genome.Within this population, only one QTL was detected on chromosome 5H between markers 12_21036 and JHI-Hv50k-1016-311034 with a LOD score of 4.93 (Figs.2C and 3A).LOD thresholds were calculated at 3.098 (α = 0.05) and 4.031 (α = 0.01).This 31 cM genetic interval corresponds to a 33.3 Mb physical interval, encompassing the previously delimited locus identified within the CI5791 × Tifang population (Fig. 4).

CI5791 genome assembly and Spt2 refinement
The CI5791 genome assembly was sequenced to a depth of 50x.Reads were assembled into 23,457 contigs (N50 of 309 kb) and scaffolded into the 12 pseudomolecule chromosomes of the Morex V3 genome assembly.Alignment of the Spt2 region from CI5791 and Golden Promise identified multiple polymorphism that could be targeted with PACE.A total of two from 41 polymorphisms targeted were successfully converted to PACE and polymorphic between CI5791 and Tifang for analysis of the remaining 14 critical recombinants.From the four susceptible critical recombinants, one delimits the Spt2 region to a single candidate gene annotated in the Morex V3 assembly as a PPR.A further four of the ten resistant critical recombinants also delimit the Spt2 region to the PPR gene.Therefore, the Spt2 interval was delimited between the markers 5H_438381417 and 5H_492358011 (~ 198 kb) by five critical recombinants (Fig. 3).

Pangenome assessment
The genomic interval delimiting Spt2 between markers 5H_438381417 and 5H_492358011 corresponds to a ~ 198 kb physical interval within the Morex V3 assembly and ranged from 177 to 266 kb within all the currently released barley genomes (Table 1).The parental lines CI5791 and Golden Promise harbor ~ 217 and ~ 200 kb physical intervals, respectively.Whereas the parental line Tifang does not currently have sequence data available.The CI5791 Spt2 region is one the longest identified to date and the Golden Promise interval remains in concordance with other Western cultivars (Table 1).Both CI5791 and Golden Promise share high collinearity and synteny with Morex with only a deletion detected in the third syntenic block of CI5791 and a small inversion detected within Golden Promise when compared to CI5791 and Morex (Fig. 5A).With the exception of Haruna Nijo, a Japanese malting line, phylogenetic analysis of the Spt2 locus reveals two main monophyletic groups separating European accessions and one American accession Morex with Asian and African accessions (Fig. 5B).

Canadian accession AAC Synergy and American accession Hockett form their own clades.
There are between one and two genes present within individual barley accessions, with a total of two unique genes present collectively across the delimited Spt2 locus in all currently released pseudochromosome level genomes (Table 1).These genes include the PPR previously annotated in the Morex V3 assembly and a lysine tRNA ligase.However, only the PPR is consistently annotated in the majority of barley lines and the remaining lysine tRNA ligase gene is present in only seven wild and landrace accessions.The PPR is present in all lines except cultivar Zangqing320 from China.When comparing the PPR between CI5791 and Golden Promise, neither gene contains exonic polymorphisms, and therefore are predicted to have conserved amino acid sequences of the translated protein (Table 2).The PPR contains polymorphism within the intronic region, however these are not predicted to alter the splice site junctions (Table 2).Lastly, the PPR contains polymorphism upstream and downstream of the gene that may affect gene expression.The lysine tRNA ligase is not present within the Spt2 delimited region in either CI5791 or Golden Promise.

Discussion
The Spt2 gene which confers hybrid susceptibility was mapped to a single locus, whereby individuals heterozygous at the Spt2 locus exhibit significantly increased susceptibility compared to either parent that are both resistant to the Ptm isolate 13IM8.3.This is the first documented case of non-transgressive segregation that supports the hybrid susceptibility model where susceptibility only manifests in the heterozygous state [28].The Spt2 locus is therefore non-transgressive as the locus was not mapped in previous mapping studies that utilized the CI5791 × Tifang recombinant inbred line (RIL) population and the same Ptm isolate 13IM8.3[29].In addition, this phenomenon is distinct from post-zygotic genetic incompatibilities such as hybrid necrosis that involves epistatic interactions between at least two loci and occur despite the absence of pathogen [26,27], as no hybrid necrosis was observed during the development of the CI5791 × Tifang RIL population [30].
The Spt2 locus was mapped to the same region within two independent populations that share CI5791 as a common parent.This raises the question of whether the same phenomena would exist in a Tifang × Golden Promise population and whether CI5791 is the main driver behind hybrid susceptibility.Due to the marker pool used on the CI5791 × Golden Promise population, the Spt2 locus encompasses a 33.predicted to encode a PPR present in the publicly available Golden Promise [32], and Morex V3 [31] genome assemblies, and the CI5791 genome assembly generated for this study.As Spt2 mapped to the same region in the CI5791 × Tifang and CI5791 × Golden Promise populations and comparative analysis of the CI5791 and Golden Promise genome sequences identified highly colinear, syntenic regions, we are confident this region represents Spt2 in both populations.
Based on function and the current body of literature, the PPR candidate gene could be implicated in the heterozygous susceptibility phenomenon.Loss-of-function and knockdown mutants of PPR proteins in Arabidopsis have previously been implicated in increased susceptibility to bacterial and fungal pathogens [33].PPR proteins are known to be found at exceptionally higher levels in plant organelles and shown to bind single stranded RNA in a similar manner to transcription activation-like effectors to DNA [34].These PPR proteins are hypothesized to be at increased levels in plant organelles due to the lack of promoters in organelle genomes and therefore rely on PPR proteins to regulate gene expression at the RNA level affecting transcription, processing, splicing, stability, editing and translation.
Heterosis has typically been explained by classical genetic models such as dominance, overdominance and epistasis of multiple genes [35].As Spt2 has been mapped to a single locus and comparative analysis determined that the PPR does not contain polymorphisms within exons that will change the primary amino acid sequence of the proteins, we therefore hypothesize that the causal gene underlying the locus results in hybrid susceptibility is due to insufficient expression, or overexpression of encoded protein/s caused by the polymorphisms in regulatory components.The PPR gene contains polymorphisms upstream, downstream and within introns between CI5791 and Golden Promise that may therefore affect the transcriptional regulation of the heterozygous alleles [36,37].These upstream, downstream and/ or intronic polymorphisms could explain differential expression of the Spt2 gene when present in a heterozygous state.We do not currently hypothesize that posttranscriptional modification of the proteins or alterations of protein complexes are affected as and the PPR is predicted to contain identical primary amino acid sequence in both the CI5791 and Golden Promise parents.
In addition, heterosis has often been associated with the genetic distance of the two parents, in that interspecies crosses exhibit increased heterosis over intraspecies crosses [35].However, there is a growing body of evidence showing that this does not hold true and heterosis can be explained by epigenetic factors in intraspecies crosses [38][39][40].For example, reports in Arabidopsis have shown that despite near identical genomes, different small interfering RNAs (siRNAs) and methylation states of each parent can produce F 1 progeny with approximately 250% increased biomass [38].Therefore, due to the fact that a single locus exhibiting limited genetic polymorphism within the PPR gene has been identified within an intraspecies cross, we also hypothesize that hybrid susceptibility could be the result of altered gene expression caused by altered siRNA, methylation states or histone modification at the Spt2 locus within heterozygous progeny.Subsequently, the identified genetic polymorphisms at the Spt2 locus are merely linked to these altered states when in the heterozygous state and not causal to the Spt2-mediated susceptibility.This altered expression of the Spt2 gene in the heterozygous state, either suppressed or increased expression could result in loss of a resistance factor or the overexpression of a susceptibility target, respectively.
Interestingly, the Spt2 locus colocalizes with the previously mapped loci NBP_QRptt5-1 [41], QRpt-5 H.2 [42], and potentially an unnamed QTL [17,43].Therefore, we propose the primary locus designation as Resistance to Pyrenophora teres 10 (Rpt10), with the secondary locus designation as Spt2 (Susceptibility to Pyrenophora teres 2).Thus, Rpt10/Spt2 may represent a resistance locus when in a homozygous state and a susceptibility locus when held in specific heterozygous states.However, further genetic analysis and gene characterization would need to be conducted to verify if the Spt2 locus definitively colocalizes with the Rpt10 locus and represents a singular Rpt10/Spt2 locus similar to the Rpt5/Spt1 locus in the barley-Ptt pathosystem [16].
With farmers increasingly relying on hybrid barley to maximize agronomic potential, discovery of the mechanisms underlying hybrid susceptibility is paramount to avoid the inadvertent release of hybrids with deleterious phenotypes.Hybrid barley is seen as a potential solution to combat stagnating yield increases, particularly in European feed lines, but could be applied to malting barley in the face of climate change [1,3].To our knowledge, this is the first documented case of non-transgressive, hybrid susceptibility within a single plant species, which could have grave consequences for hybrid cultivars if not addressed prior to release.Additional questions arise from the mechanism by which this hybrid susceptibility functions and whether genetic or epigenetic polymorphisms drive this mechanism.Elucidating this hybrid susceptibility mechanism will allow the identification of suitable and unsuitable parents in hybrid barley breeding programs and the release of both high quality and resistant hybrids.

Conclusion
A rare non-transgressive hybrid susceptibility locus designated Spt2 was mapped with high resolution to a single candidate gene on chromosome 5H of barley using multiple rounds of mapping in a CI5791 × Tifang population.This non-transgressive hybrid susceptibility locus was also identified in a second CI5791 x Golden Promise population that shared CI5791 as a common parent.This single Spt2 candidate gene was predicted to encode a PPR, a class of genes that had been previously implicated in susceptibility in Arabidopsis to bacterial and fungal pathogens.The Spt2 locus was previously mapped as a resistance locus and therefore may function under a resistance mechanism when held in a homozygous state and a susceptibility locus when held in the heterozygous state.Experiments to validate this gene using reverse genetic approaches have been initiated.This study provides a base to further characterize and validate the Spt2 locus and provides additional knowledge to understand the molecular mechanisms underlying hybrid susceptibility and vigor.

Plant materials
To reduce environmental variability all barley seedlings were grown in Argu soil in environmentally controlled growth chambers (Conviron, Winnipeg, Canada) under a 12 h light/12 h dark cycle, at 21 °C.The F 2 seedlings used for genetic mapping of Spt2 were derived from CI5791 × Tifang or CI5791 × Golden Promise crosses.CI5791 is a two-row Ethiopian landrace, Tifang is a six-row Chinese landrace, and Golden Promise is a Scottish two-row malting cultivar.The F 1 individuals were self-fertilized to generate F 2 seed and individual F 2 seed were planted in cone-tainers for the first two rounds of mapping, and 96-cell trays for the final round of mapping using the CI5791 × Tifang population and the CI5791 × Golden Promise population.

Pathogen isolates and phenotypic screening
One-week post planting of each of the F 2 populations, dried agar plugs of the Ptm isolate 13IM.3 were placed on plates of V8-PDA media and allowed to grow for approximately seven days in the dark before exposing them to a 24 h light/24 h dark cycle to induce sporulation.The Ptm isolate 13IM.3 was collected from Blackfoot, Idaho in 2013 [44].After sporulation was induced the plates were flooded with ~ 10 ml of ddH 2 0 and scraped using a sterile inoculating loop.The conidia spores collected in the water suspension were counted under a microscope using a 5 µl droplet and adjusted to ~ 2,000 spores/ ml with one drop (~ 30 µl) of Tween 20 surfactant per 50 ml.Plants were spray inoculated at approximately 20 psi until runoff at the second leaf stage which was ~ 14 days post planting under growth chamber conditions, placed in a mist chamber with 24 h light conditions and misted to maintain 100% relative humidity.After 24 h in the mist chamber, the inoculated seedlings were allowed to air dry and moved back to the growth chamber set at a 12 h light/12 h dark cycle, and 21 °C.Seven days post inoculation plants were phenotypically scored using the 1-5 SFNB rating scale described by [45].For a disease reaction score of 1 consisted of pinpoint lesions; a disease reaction score of 2 consisted of pinpoint lesions with small amounts of chlorosis/necrosis; a disease reaction score of 3 consisted of lesions 2-3 mm in size with little to no coalescence; a disease reaction score of 4 consisted of lesions large than 3 mm with moderate coalescence of less than 70% of leaf area; a disease reaction score of 5 consisted of lesions with coalescence greater than 70% of the leaf area.Individuals were considered resistant when less than 3 on the rating scale and susceptible when 3 or above on the rating scale.Phenotyping could not be repeated due to the genetic nature of the Spt2-mediated hybrid susceptibility which required mapping with F 2 individuals.Successive steps of higher resolution mapping and increased marker saturation were performed utilizing additional F 2 progeny from the CI5791 × Tifang cross.Each step of genetic mapping was conducted independently with exception of the last rounds within the CI5791 × Tifang high-resolution population which utilized DNA from individuals identified in the previous screen as well as the same phenotypic data for further marker saturation in the regions identified with critical recombination events.

DNA extraction for genotyping
Tissue from individual seedlings were collected in 96-deep well plates prior to inoculation and DNA extracted for 9K derived initial and 50K derived highresolution mapping using PCR genotyping-by-sequencing (PCR-GBS) as described by [16] and [46] using the Ion Torrent PGM platform.For the final round of highresolution mapping and low-resolution mapping with the CI5791 × Golden Promise population, the leaf tissue was collected in sample plates, frozen at -80 °C, lyophilized and DNA extracted using the oKtopure™ automated DNA extraction platform following the manufacturer's instructions.

CI5791 × Tifang 9 K derived genetic mapping
For initial genetic mapping using 106 CI5791 × Tifang F 2 individuals (representing 212 recombinant gametes), a custom panel of 365 SNPs derived from the 9K Illumina SNP data [47] was utilized (Supplemental File 1).The SNP markers utilized to develop the custom PCR-GBS panel for amplicon sequencing were derived from the barley 9K iSelect SNP platform data [47].The 365 SNPs were selected based on polymorphism between CI5791 and Tifang as well as uniform distribution across the barley genome.Primers were designed from the source sequences extracted from the T3/Barley database (https://barley.triticeaetoolbox.org) to produce ~ 125 bp amplicons over the targeted SNPs.Primers were tagged with CS1 and CS2 adapters (Fluidigm, San Francisco CA) for forward and reverse primers, respectively.Each primer was tested individually for amplification and primers pooled to generate ten multiplex pools.Subsequently, PCR-GBS was performed as described by [16] and [46] on the Ion Torrent PGM platform.Reads were trimmed by 22nt in CLC Genomics Workbench to remove adapters.Trimmed reads were aligned to the reference amplicon panel using bwa-mem [48], converted and sorted to BAM files using samtools [49] and SNPs called using the Genome Analysis Toolkit Haplot-ypeCaller tool [50,51].SNPs were filtered using vcftools [52] using the following filters --minQ 30 --minDP 3 and --max-missing 0.5.MapDisto 2.1.7[53] was used for genetic map construction and file export for QTL mapping in QGene 4.4.0 [54] using the composite interval mapping algorithm and default parameters for selecting cofactors (Supplemental File 2).LOD thresholds at α level = 0.05 and α level = 0.01 were calculated using a 1000 permutation test.LOD scores and significance thresholds were parsed for visualization using ggplot2 within R 4.1.2.

CI5791 × Tifang 50K derived high-resolution mapping
Following initial mapping, both parental lines were subjected to the barley 50K iSelect genotyping platform [55].Molecular markers within the region delimited by the initial genetic mapping were analyzed for polymorphisms between CI5791 and Tifang.The analysis identified 22 polymorphic SNPs (Supplemental File 3) based extracted from the T3/Barley database (https://barley.triticeaetoolbox.org).Primers for the RAD-GBS panel were designed using Primer3-Plus within Geneious Prime® (https://www.geneious.com),and checked for specificity using BLAST on the EnsemblPlants [56] using the barley genome.Multiplex robustness was optimized by detecting predicted primer dimer and multiplex ability using MFE-Primer [57].The PCR-GBS and SNP calling pipeline followed the same protocol as the initial mapping except reads were trimmed using Trimmomatic [58].High-resolution QTL mapping was performed with 340 CI5791 × Tifang F 2 individuals (680 recombinant gametes) in the same manner as the initial mapping described above (Supplemental File 4).

CI5791 × Tifang exome capture derived high-resolution mapping
Five lines exhibiting recombination within the delimited region were selected for exome capture.SeqCap EZ HyperCap exome capture [59] was performed as per the manufacturer's instructions (Roche Sequencing Solutions, Pleasanton, CA, USA) with the following adjustments.Starting DNA concentration was increased from the recommended 100 ng to 300 ng per sample based on Hoescht DNA quantification and pooled DNA concentration was increased from 1 µg to 2 µg.The final purified pool was sequenced on a single flow cell on the Illumina NextSeq®500 sequencing platform.A total of 46 polymorphic SNPs (Supplemental file 5) were identified from the exome capture sequencing data and oligonucleotide primer pairs were designed as previously described for the 9K SNP source sequences with the following adjustments.The 5' termini of the primers were tagged with M13 and P1/B sequences to facilitate barcoding.DNA from the 340 F 2 individuals from the 50K derived high-resolution mapping and an additional 269 F 2 individuals were utilized for a total of 609 CI5791 × Tifang F 2 individuals representing 1,218 recombinant gametes.Sequencing was performed using an Ion PI™ chip on the Ion Proton™ system platform.Individual fastq files were parsed using fastq-grep command due to the unequal barcode lengths used to increase the number of samples in the multiplex sequencing library.SNP calling preceded as described in 50K derived high-resolution mapping and QTL mapping was performed in the same manner as 9K derived initial mapping as described above (Supplemental file 6).CI5791 × golden Promise genotype-by-multiplex sequencing mapping A total of 83 CI5791 × Golden Promise F 2 individuals were screened for disease response as described in biological materials and phenotyping.After DNA extraction, accessions were genotyped using a GMS marker panel [60] using Illumina sequencing.SNP calling preceded as described in exome derived high-resolution mapping of CI5791 × Tifang except the reads were trimmed using fastp [61].QTL mapping was performed in the same manner as 9K derived initial mapping as described above (Supplemental File 7).

CI5791 genome assembly
The barley accession CI5791 was subjected to whole genome sequencing for genome assembly.Briefly, barley was grown to the three-leaf stage and dark treated for 24 h.Leaves were cut, and 50 g of young leaf tissue was weighed and wrapped in aluminum foil and immediately submerged in liquid nitrogen.Tissue was subsequently stored at -80 o C. High molecular weight DNA was extracted, and sequencing performed using one SMRT Cell 8M in Continuous Long Read mode of the PacBio Sequel II System at the University of Arizona Genomics Institute.Raw reads were assembled into contigs using Canu 2.2.1 [62], and scaffolded to the Morex V3 reference genome assembly [28].

Spt2 refinement using CI5791 and Golden Promise polymorphisms
Fourteen critical recombinants were identified from the 609 CI5791 × Tifang F 2 individuals screened within the delimited 1.2 cM (~ 1.5 Mb) Spt2 region that could be utilized for additional marker saturation based on identified polymorphisms.As all identified polymorphisms were exhausted from previous genotyping platforms, the CI5791 genome assembly and the publicly available Golden Promise genome assembly were utilized to identify novel polymorphism to further saturate the region with genetic markers.To identify polymorphisms between CI5791 and Golden Promise, 500 kb intervals of the Spt2 locus were aligned using MAFFT [63].SNPs and 1 bp indels between CI5791 and Golden Promise were targeted for PACE (3CR Bioscience, Harlow, UK) primer design within Geneious Prime® 2023.2.1 (https://www.geneious.com).Two polymorphisms from the 41 identified were targeted and successfully converted to PACE genotyping assays (Supplemental File 8).Primer designs followed 3CR Bioscience guidance, while minimizing Tm primer differences to less than 1 °C and avoiding homopolymer runs.Primer specificity was determined using EnsemblPlants [56] BLAST tool and the Morex V3 genome assembly.Oligonucleotides were synthesized (Sigma-Alrich, St. Loius, MO, USA) and diluted to 100 µM stocks.Primer concentrations followed manufacturers recommendations with working primer assay stocks created by combining 12 µl of each forward primer, 30 µl of the common reverse primer, and 46 µl of water.Assay mastermixes were produced by combining a total of 5.0 µl standard ROX PACE mastermix (3CR Bioscience, Harlow, UK) and 0.14 µl of working primer assay per sample.Cycling conditions were optimized by performing 10 µl reactions containing 5.0 µl assay mastermix and 5.0 µl of parental DNA and two non-template water controls (NTCs) in 384-well plate format on a CFX384 qPCR thermocycler (Bio-Rad Laboratories, Hercules, CA, USA).Cycle number was optimized by assessing every two cycles between 26-36x cycles post touchdown of 10 cycles (61-55 °C, − 0.6 °C/cycle).Assays were deemed successful if two distinct homozygous clusters were obtained with one group consisting of CI5791 samples and the second group consisting of Golden Promise and Tifang samples.The successfully converted assays were subsequently performed on the 14 CI5791 × Tifang F 2 individuals critical recombinants identified in the previous round using identical conditional with two samples of each parent, two CI5691 × Tifang F 1 samples and two NTC samples as controls.All results were analyzed in CFX Maestro 1.1 (Bio-Rad Laboratories) (Supplemental File 9).Pangenome Assessment.

Fig. 3
Fig. 3 Genetic mapping of the Spt2 locus within the CI5791 × Tifang population.(A) Sequential mapping utilizing increased marker saturation and number of recombinant gametes corresponding to 9K, 50K, and exome capture sequencing and whole genome alignment derived marker panels.A representative set of F 2 progeny are shown at each mapping stage with the specific accession ID indicated below the chromosome, whether the accession was resistant (R) or susceptible (S) and the total number of recombinant gametes (RGs) screened are indicated above.The parental sequences are shown in yellow (CI5791) or blue (Tifang), and heterozygous sequences in green.Black crosses indicate that recombination occurred between the two markers but do not indicate approximate location.The delimited region is marked as red along the chromosome and marker names in bold.Note that progeny numbers were reset from the second round of mapping.(B) Physical interval within Morex V3 with genes shown as magenta arrows

Fig. 4 1
Fig. 4 Genetic mapping of the Spt2 locus within the CI5791 × Golden Promise population.(A) A single F 2 population using a 50 K derived marker panel in comparison to the CI5791 × Tifang Spt2 mapping population.A representative set of F 2 progeny are shown at each mapping stage with the specific accession ID indicated below the chromosomes, whether the accession was resistant (R) or susceptible (S) and the total number of recombinant gametes (RGs) are indicated above.The parental sequences are shown in yellow (CI5791) or blue (Golden Promise), and heterozygous sequences in green.Black crosses indicate that recombination occurred between the two markers but do not indicate approximate location.The delimited region is marked as red along the chromosome and marker names in bold.(B) The refined mapping of the Spt2 locus using the CI5791 × Tifang F 2 mapping population.(C) Physical interval within Morex V3 with genes shown as magenta arrows

3 Fig. 5
Fig. 5 Pangenome comparisons of the Spt2 region.(A) Mauve alignment of the Spt2 locus in CI5791 (top) and Golden Promise (middle), against the reference genome assembly of Morex V3 (bottom).Different colors indicate different syntenic colinear blocks.Blocks above and below the central line indicate sense and antisense.(B) Phylogenetic tree using the Spt2 locus from all 25 released barley genomes using MAFFT for alignment and RAxML for tree construction